Hepatocyte-specific deletion of Bis causes senescence in the liver without deteriorating hepatic function.

Bcl-2-interacting cell death suppressor (BIS), also called as BAG3, regulates numerous physiological processes, such as apoptosis, protein quality control, and senescence. Whole-body Bis-knockout (KO) mice exhibit early lethality following cardiac and skeletal muscle dysfunction. The first attempt to generate organ-specific knockout mice resulted in constitutive or inducible heart-specific Bis-knockout mice, which exhibited cardiac dilation and underwent premature death. Here, we generated hepatocyte-specific Bis-knockout (Bis-HKO) mice and found no abnormalities in metabolic function and survival. However, depletion of HSPB8 and accumulation of p62 indicated impaired autophagy in Bis-HKO livers. Interestingly, the number of peroxisomes wrapped by phagophore membranes increased as evidenced by transmission electron microscopy analysis, indicating defects in the progression of pexophagy. In addition, increased dihydroethidine intensities and histone H3 K9me3-positive nuclei indicated increased oxidative stress and senescence induction in Bis-HKO livers. Mechanistically, p27 was upregulated in Bis-HKO livers. In SNU368 hepatocellular carcinoma cells, BIS depletion led to p27 upregulation, and increase in histone H3 K9me3 levels and senescence-associated ß-galactosidase staining; therefore, reproducing the in vivo senescence phenotype. Despite the observation of no metabolic abnormalities, BIS depletion led to defective autophagy, increased oxidative stress, and senescence in Bis-HKO livers. Collectively, our results suggest a role for BIS in maintaining liver regeneration potential under pathological conditions.

Induction of BIS Protein During Astroglial and Fibrotic Scar Formation After Mitochondrial Toxin-Mediated Neuronal Injury in Rats.

B cell leukemia/lymphoma-2 (Bcl-2)-interacting death suppressor (BIS), also identified as Bcl-2-associated athanogene 3 (BAG3), has been reported to be upregulated in reactive astrocytes after brain insults. The present study was designed to further substantiate the involvement of BIS protein in the astroglial reaction in the striatum of rats treated with the mitochondrial toxin, 3-nitropropionic acid. Weak constitutive immunoreactivity for BIS was observed in astrocytes in the control striatum, whereas its expression was upregulated, along with that of nestin, in the lesioned striatum. In the lesion core, where astrocytes are virtually absent, BIS/nestin double-labeled cells were associated with the vasculature and were identified as perivascular adventitial fibroblasts. By contrast, BIS/nestin double-labeled cells in the perilesional area were reactive astrocytes, which were confined to the border zone contributing to the formation of the astroglial scar; this was evident 3 days post-lesion and increased thereafter progressively throughout the 28-day experimental period. At the ultrastructural level, BIS protein was diffusely localized throughout the cytoplasm within the stained cells. Collectively, our results demonstrate the phenotypic and functional heterogeneity of BIS-positive cells in the lesioned striatum, suggesting the involvement of BIS in the formation of astroglial scar and its potential role in the development of fibrotic scar after brain insults.

14-3-3ζ targeting induced senescence in Hep-2 laryngeal cancer cell through deneddylation of Cullin1 in the Skp1-Cullin-F-box protein complex.

OBJECTIVES: Despite of the aberrant expression of 14-3-3ζ in head and neck squamous cell carcinoma (HNSCC), little is known about the role of 14-3-3ζ in the regulation of senescence in HNSCC. This study was performed to investigate whether 14-3-3ζ is implicated in senescence evasion of Hep-2 laryngeal cancer cells. METHODS: The expression of 14-3-3ζ was suppressed using RNA interference strategy. Senescence induction was determined by senescence-associated ß-galactosidase staining and the numbers of promyelocytic leukaemia nuclear body. Real-time PCR, western blotting and immunohistochemistry were applied for the expression of corresponding proteins. Xenograft experiment was performed to show in vivo effect of 14-3-3ζ silencing on tumour growth. RESULTS: 14-3-3ζ silencing significantly induced senescence phenotypes via 27 accumulations. Subsequently, we demonstrated that p27 accumulation is linked to inactivation of SCFSkp2 complex activity, probably due to the deneddylation of cullin-1 (Cul-1) as follows. (a) Neddylated Cul-1 is decreased by 14-3-3ζ silencing. (b) Blocking neddylation using MLN4924 reproduces senescence phenotypes. (c) Knockdown of CSN5, which functions as a deneddylase, was shown to restore the senescence phenotypes induced by 14-3-3ζ depletion. Finally, we demonstrated that 14-3-3ζ depletion effectively hindered the proliferation of Hep-2 cells implanted into nude mice. CONCLUSION: 14-3-3ζ negatively regulates senescence in Hep-2 cells, suggesting that 14-3-3ζ targeting may serve to suppress the expansion of laryngeal cancer via induction of senescence through the Cul-1/SCFSkp2 /p27 axis.

Mitochondrial glutamine metabolism via GOT2 supports pancreatic cancer growth through senescence inhibition.

Cellular senescence, which leads to a cell cycle arrest of damaged or dysfunctional cells, is an important mechanism to restrain the malignant progression of cancer cells. Because metabolic changes underlie many cell-fate decisions, it has been suggested that cell metabolism might play key roles in senescence pathways. Here, we show that mitochondrial glutamine metabolism regulates senescence in human pancreatic ductal adenocarcinoma (PDAC) cells. Glutamine deprivation or inhibition of mitochondrial aspartate transaminase (GOT2) results in a profound induction of senescence and a suppression of PDAC growth. Glutamine carbon flow through GOT2 is required to create NADPH and to maintain the cellular redox state. We found that elevated reactive oxygen species levels by GOT2 knockdown lead to the cyclin-dependent kinase inhibitor p27-mediated senescence. Importantly, PDAC cells exhibit distinct dependence on this pathway, whereas knockdown of GOT2 did not induce senescence in non-transformed cells. The essentiality of GOT2 in senescence regulation of PDAC, which is dispensable in their normal counterparts, may have profound implications for the development of strategies to treat these refractory cancers.

14-3-3ß Depletion Drives a Senescence Program in Glioblastoma Cells Through the ERK/SKP2/p27 Pathway.

The induction of senescence in cancer cells has recently been implicated as a mechanism of tumor regression in response to various modes of stress. 14-3-3 proteins are conserved scaffolding molecules that are involved in various cellular functions. Among the seven isoforms, 14-3-3ß is specifically expressed in astrocytoma in correlation with the malignancy grade. We investigated the possible role of 14-3-3ß in the regulation of senescence induction in A172 glioblastoma cells. The knockdown of 14-3-3ß by specific small interfering RNA resulted in a significant change in cellular phenotypes and an increase in cells staining positive for senescence-associated ß-galactosidase. Western blotting of the 14-3-3ß-depleted A172 cells revealed increased p27 expression and decreased SKP2 expression, while the expression of p53 and p21 was not altered. Subsequently, we demonstrated that ERK is a key modulator of SKP2/p27 axis activity in 14-3-3ß-mediated senescence based on the following: (1) 14-3-3ß knockdown decreased p-ERK levels; (2) treatment with U0126, an MEK inhibitor, completely reproduced the senescence morphology as well as the expression profiles of p27 and SKP2; and (3) the senescence phenotypes induced by 14-3-3ß depletion were considerably recovered by constitutively active ERK expression. Our results indicate that 14-3-3ß negatively regulates senescence in glioblastoma cells via the ERK/SKP2/p27 pathway. Furthermore, 14-3-3ß depletion also resulted in senescence phenotypes in U87 glioblastoma cells, suggesting that 14-3-3ß could be targeted to induce premature senescence as a therapeutic strategy against glioblastoma progression.